Archaeal pyrrolysyl trna synthetases for orthogonal use

Abstract

The invention relates to archaeal pyrrolysyl tRNA synthetases lacking a nuclear localization signal and / or comprising a nuclear export signal. The invention also relates to polynucleotides encoding said pyrrolysyl tRNA synthetases, eukaryotic cells com- prising said polynucleotide and tRNA acylated by the pyrrolysyl tRNA synthetase or a polynucleotide encoding such tRNA, methods utilizing said cells for preparing polypeptides comprising unnatural amino acid residues, and kits useful in said methods.

Description

1 Archaeal pyrrolysyl tRNA synthetases for orthogonal use FIELD OF THE INVENTION The invention relates to archaeal pyrrolysyl tRNA synthetases lacking a nuclear locali- zation signal and / or comprising a nuclear export signal, polynucleotides encoding said pyrrolysyl tRNA synthetase, eukaryotic cells comprising said polynucleotide and tRNA acylated by the pyrrolysyl tRNA synthetase or a polynucleotide encoding such tRNA, methods utilizing said cells for preparing polypeptides comprising unnatural amino acid residues, and kits useful in said methods. BACKGROUND OF THE INVENTION The ability to visualize biomolecules within living specimens by engineered fluores- cence tags or other labels which allow imaging has become a major tool in modern biotechnology, cell biology, and life sciences. A major challenge common to these la- bel-based imaging techniques is to genetically encode a labeling site that is, ideally, as small as possible. Genetic code expansion resulting in translational modification of proteins by direct ge- netic encoding of unnatural amino acids, in particular using stop codon suppression, by means of a tRNA / aminoacyl tRNA synthetase (tRNA / RS) pair that is orthogonal to the host machinery offers exquisite specificity, freedom of placement within the target pro- tein and minimal structural change. This approach has meanwhile been used to genet- ically encode several unnatural amino acid residues of interest. For instance, engi- neered Methanococcus jannaschii tRNA / tyrosyl tRNA synthetase, E. coli tRNA / leucyl tRNA synthetase as well as Methanosarcina mazei and M. barkeri tRNA / pyrrolysyl tRNA synthetase pairs have been used to genetically encode a variety of functionalities in polypeptides (Chin et al., J Am Chem Soc 124:9026, 2002; Chin et al., Science 301:964, 2003; Nguyen et al, J Am Chem Soc 131:8720, 2009, Yanagisawa et al., Chem Biol 15:1187, 2008). Up to now, more than 200 different unnatural amino acids (for review see e.g. Liu et al., Annu Rev Biochem 83:379-408, 2010; Lemke, ChemBi- oChem 15:1691-1694, 2014) have been incorporated with residue precision. The present inventors and others have recently shown that unnatural amino acids con- taining strained alkynyl or strained alkenyl groups can be encoded in living mammalian cells in response to the amber codon by means of the tRNA / pyrrolysyl tRNA synthe- tase pair originating from Methanosarcina mazei (Plass et al., Angew Chem Int Ed Engl 51(17):4166-4170, 2012; WO 2012 / 104422). Polypeptides which comprise unnatural amino acid residues carrying reactive groups such as strained alkynyl, strained alkenyl or norbornenyl groups can be used for ultrafast and bioorthogonal click reactions, such as strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) and strain promoted alkyne-azide cycloaddition (SPAAC), with tetrazines or azides, respec- tively. Dyes functionalized with tetrazines have previously been used in such click reac- tions to label either surface proteins or cytoskeletal proteins in mammalian cells for very high resolution imaging methods (Nikic et al., Angew Chem Int Ed Engl 53(8): 2245-2249, 2014; WO 2015 / 107064; Uttamapinant et al., J Am Chem Soc 137(14):4602-4605, 2015). Among the biggest issues of genetic code expansion in general, and thus in particular for high resolution imaging methods based thereon, is the competition of stop codon suppression with the host's internal translation termination machinery which leads to limited efficiency in labeling less-abundant proteins inside cells. Many approaches have been explored to address this key issue of genetic code expansion in eukaryotes, in- cluding promoter engineering, better evolution of the RSs, release-factor engineering and multi-chaining of tRNAs, to just name a few (for review see, e.g., Chin et al., Annu Rev Biochem 83:379-408, 2014). Despite these efforts, there is still a high demand for strategies which improve efficien- cy of genetic code expansion in eukaryotic cells (and thus the amount of target poly- peptide comprising unnatural amino acid residues that is expressed by the cell and can be used for labeling and imaging purposes) so as to allow for efficient labeling, even of polypeptides of low abundance, and the use of super-resolution microscopy (SRM). It was therefore an object of the present invention to address this challenge. SUMMARY OF THE INVENTION The inventors identified a sequence within archaeal pyrrolysyl tRNA synthetases that can act as a nuclear localization signal in eukaryotic cells. The inventors showed that the efficiency of genetic code expansion based on archaeal pyrrolysyl tRNA synthetase can be increased if the amino acid sequence of the synthetase is modified such that it is not directed to the nucleus. To this end, the nuclear localization signal can be re- moved from the synthetase or can be overridden by introducing a suitable nuclear ex- port signal. The inventors assume that mislocation of archaeal pyrrolysyl tRNA synthetases ex- pressed within eukaryotic cells to the nucleus limits the efficiency of genetic code ex- pansion based on such synthetases because it limits the amount of synthetase availa- ble in the cytoplasm where translation takes place. It is believed that during translation an unnatural amino acid is more likely to be incorporated into a growing polypeptide 3 chain if, in the cytoplasm of the cell, in particular at the ribosomes, there is a high con- centration of archaeal pyrrolysyl tRNA synthetase and a high concentration of the tRNA that is acylated by the synthetase with the unnatural amino acid. Accordingly, the present invention relates to an archaeal pyrrolysyl tRNA synthetase (PyIRS) that lacks a nuclear localization signal (NLS) and / or comprises a nuclear ex- port signal (NES). The present invention also provides polynucleotides encoding a PyIRS of the invention. The present invention further provides a combination of polynucleotides comprising at least one polynucleotide encoding a PyIRS of the invention and at least one polynucle- otide encoding a tRNAPyl, wherein the tRNAPyl is a tRNA that can be acylated by said PyIRS. The present invention also relates to a eukaryotic cell, preferably a mammalian cell, comprising (i) a polynucleotide sequence that encodes a PyIRS of the invention and (ii) a tRNAPyl that can be acylated by said PylRS or a polynucleotide sequence that en- codes such tRNAPyl. The polynucleotide sequence encoding the tRNAPyl may be locat- ed on the polynucleotide encoding the PyIRS or on a separate polynucleotide. Expedi- ently, the eukaryotic cell is capable of expressing the PyIRS and, if applicable, the tRNAPyl. The present invention also relates to a method for preparing a target polypeptide com- prising one or more than one unnatural amino acid (UAA) residue, wherein the method comprises: (a) providing a eukaryotic cell of the present invention comprising: (i) a PylRS of the invention, (ii) a tRNA (tRNAPyl), (iii) an UAA or a salt thereof, and (iv) a polynucleotide encoding the target polypeptide, wherein any position of the target polypeptide occupied by an UAA residue is encoded by a codon that is the reverse complement of the anticodon comprised by the tRNAPyl; and wherein the PyIRS (i) is capable of acylating the tRNA (ii) with the UAA or the salt of (iii); and (b) allowing for translation of the polynucleotide (iv) by the eukaryotic cell, thereby producing the target polypeptide. The present invention also relates to a method for preparing a polypeptide conjugate comprising: 4 (a) preparing a target polypeptide comprising one or more than one UAA residue using a method of the present invention; and (b) reacting the target polypeptide with one or more than one conjugation partner molecule such that the conjugation partner molecules bind covalently to the UAA residue(s) of the target polypeptide. The present invention further relates to kits comprising a polynucleotide or combination of polynucleotides or eukaryotic cell of the present invention. According to one embod- iment, the invention provides a kit comprising at least one UAA, or a salt thereof, and a polynucleotide encoding a PyIRS of the invention. According to another embodiment, the invention provides a kit comprising at least one UAA, or a salt thereof, and a eukar- yotic cell of the present invention. The kit may further comprise a tRNA Pyl that can be acylated by the PyIRS, or a polynucleotide sequence encoding such tRNAPyl. The tRNAPyl-encoding polynucleotide sequence can be located on the polynucleotide en- coding the PyIRS of the invention or on a separate polynucleotide. The PyIRS of the invention is capable of acylating the tRNAPyl with the UAA, or salt thereof. Such kits are useful for expressing a target polypeptide having one or more than one UAA residue within a eukaryotic cell. Such kits may thus further comprise instructions for expressing a target polypeptide having one or more than one UAA residue within a eukaryotic cell, e.g. using a method of the present invention. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the structures of UAAs 1 and 2; and HEK293T cells transfected with either tRNAPyl / PyIRSAF (a) or tRNAPyl / NES-PyIRSAF (b), left panel: stained with Hoechst 33342, center panel: immunostained with polyclonal rat anti-PyIRS antibody + goat anti-rat IgG(H+L) Alexa Fluor 594 conjugate, right panel: merge. Figure 2 shows COS-7 cells transfected with either tRNAPyl / PyIRSAF (a) or tRNAPyl / NES-PyIRSAF (b), left panel: stained with Hoechst 33342, center panel: im- munostained with polyclonal rat anti-PyIRS antibody + goat anti-rat IgG(H+L) Alexa Fluor 594 conjugate, right panel: merge. Figure 3 shows HEK293T cells transfected with either tRNA Pyl / PylRS (a) or tRNAPyl / NES-PyIRSAF (b), left panel: stained with Hoechst 33342, center panel: fluores- cence in situ hybridization (FISH) with tRNAPyl, right panel: merge. Figure 4 shows COS-7 cells transfected with either tRNAPyl / PyIRSAF (a) or tRNAPyl / NES-PyIRSAF (b), left panel: stained with Hoechst 33342, center panel: fluores- cence in situ hybridization (FISH) with tRNAPyl, right panel: merge. Figure 5 shows the results of a flow cytometry analysis of HEK293T cells co- transfected with the iRFP-GFPY39TAG amber suppression reporter and one of the amber suppression pairs: tRNAPyl / PyIRSAF and tRNAPyl / NES-PyIRSAF. For each amber sup- pression pair, a transfected sample without an UAA (left) and with added BOC (right) is shown. The percentage of amber suppressing cells (gate "iRFP,GFP") is calculated based on the total transfected population (sum of cells in gates "iRFP", "iRFP,GFP" and GFP"). An additional gate with the percentage of bright double positive cells ("Bright DPs") is also shown. Figure 6 shows the results of a flow cytometry analysis of HEK293T cells co- transfected with the iRFP-GFPY39TAG amber suppression reporter and either tRNAPyl / PyIRSAF or tRNAPyl / NES-PyIRSAF to assess the amber suppression efficiency of PyIRSAF in the presence of UAA 1 (left), and the amber suppression efficiency of NES- PyIRSAF in the presence of UAA 1 (center) or in the absence of an UAA (right). Figure 6 shows the accumulated data of a titration over different DNA concentrations described in example 3 below. Figure 7 summarizes the change in the number of GFP fluorescent HEK293T cells (categorized as "dim DPs", "bright DPs" and "very bright DPs") as observed by flow cytometry in cell samples co-transfected with varying amounts of the amber suppres- sion reporter iRFP-GFP Y39TAG (ranging from 100-500 ng plasmid DNA per well) and either tRNAPyl / PyIRSAF (reference) or tRNAPyl / NES-PyIRSAF, and cultured in the pres- ence of a low concentration (50 µM, left graph) or high concentration (250 µM, right graph) of UAA 1. See also example 3. Figure 8(a) shows a schematic representation of the Click-PAINT method, wherein a polynucleotide sequence encoding a polypeptide of interest having an amino acid resi- due encoded an amber codon ("POI(TAG)"), is expressed in eukaryotic (e.g., mamma- lian) cells co-transfected with the amber suppression pair tRNAPyI / NES-PyIRSAF in the presence of an UAA comprising a trans-cyclooctenyl group (e.g. UAA 2); the expressed polypeptide of interest (POI) comprising the UAA incorporated at the amber encoded position is subjected to a two-step labeling reaction, wherein a tetrazine-coupled dock- ing DNA strand is chemically coupled to the UAA-derived amino acid residue of the POI by a SPIEDAC reaction and second, a complementary imager strand conjugated with a dye, is added to the cells. Figure 8 further shows the fluorescence signal of the fused mOrange protein for the vimentinN116→2-mOrange construct used as a control for 6 protein expression in HEK293T cells cotransfected with pVimentin N116TAG-PSmOrange and tRNAPyl / NES-PylRSAF (b); DNA-PAINT-based SRM of HEK293T cells cotransfect- ed with tRNAPyl / NES-PylRSAF and either pVimentinN116TAG-PSmOrange (c) or pGFPN149TAG-Nup153 (d) and expressing a vimentin–mOrange fusion (c) or GFP- Nup153 fusion (d) with UAA 2 incorporated at the amber encoded position, wherein the fusion proteins are labeled at the UAA 2-derived amino acid reside using the Click- PAINT protocol described herein (scale bar in zoomed images of nuclear pores is 100 nm). Figure 9 shows the results of a flow cytometry analysis of Sf21 cells co-transfected with the mCherry-GFPY39TAG amber suppression reporter and one of the amber suppression pairs: tRNAPyl / PyIRSAF or tRNAPyl / NES-PyIRSAF. For each amber suppression pair transfected samples without UAA and with different concentrations of added UAA 1 are shown. Each dot plot is divided into four sections: the upper left section shows "mCher- ry only" cells which expressed mCherry (i.e. were successfully transfected) but not GFP (i.e. were unable to suppress the amber stop codon in GFPY39TAG); the upper right section shows "double positives" cells which expressed both mCherry and GFP (i.e. which successfully incorporated UAA 1 into GFP); and the lower left section shows "double negatives" cells (i.e. which were not successfully transfected). See also exam- ple 6. Figure 10 shows the percentage of "double positives" relative to the total number of transfected cells, for the different UAA 1 concentrations in the Sf21 cell samples de- picted in Figure 9. See also example 6. Figure 11 shows the geometric mean of the GFP signal in the "double positives" for the different UAA 1 concentrations in the Sf21 cell samples depicted in Figure 9. See also example 6. DETAILED DESCRIPTION OF THE INVENTION Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, how- ever, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The present invention provides an archaeal PyIRS that (a) lacks an NLS, or (b) com- prises a NES, or (c) both of (a) and (b). Pyrrolysyl tRNA synthetase (PyIRS) is an aminoacyl tRNA synthetase (RS). RSs are enzymes capable of acylating a tRNA with an amino acid or amino acid analog. Expe- diently, the PyIRS of the invention is enzymatically active, i.e. is capable of acylating a tRNA (tRNAPyl) with a certain amino acid or amino acid analog, preferably with an UAA or salt thereof. The term "archaeal pyrrolysyl tRNA synthetase" (abbreviated as "archaeal PylRS") as used herein refers to a PyIRS, wherein at least a segment of the PyIRS amino acid sequence, or the entire PyIRS amino acid sequence, has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at last 99%, or 100% sequence identity to the amino acid sequence of a naturally occurring PyIRS from an archaeon, or to the amino acid sequence of an enzymatically active fragment of such naturally occurring PylRS. In particular embodiments of the invention, the archaeon is a Methanosarcina species, for example M. mazei or M. barkeri. According to a preferred embodiment of the inven- tion, the archaeon is M. mazei. According to a further preferred embodiment of the in- vention, the archaeon is M. barkeri. The PyIRS of the present invention may comprise a wildtype or mutant archaeal PyIRS, or an enzymatically active fragment thereof. Mutant archaeal PylRSs differ from the corresponding wildtype PylRSs in comprising additions, substitutions and / or deletions of one or more than one amino acid residue. Preferably, these are modifications which improve PylRS stability, alter PylRS sub- strate specificity and / or enhance PyIRS enzymatic activity. For example, the mutant archaeal PyIRS is a mutant as described in Yanagisawa et al., Chem Biol 2008, 15:1187, or EP2192185. According to a particular embodiment, the PyIRS of the invention comprises M. mazei wildtype PyIRS, or an enzymatically active fragment thereof. The amino acid sequence of wildtype M. mazei PylRS is set forth in SEQ ID NO:1. SEQ ID NO:1 MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARAL 60 RHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLE 120 NTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMS 180 APVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERE 240 NYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPM 300 LAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLE 360 8 SIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGA 420 GFGLERLLKVKHDFKNIKRAARSESYYNGISTNL 454 According to another particular embodiment, the PyIRS of the invention comprises a mutant M. mazei PylRS, or an enzymatically active fragment thereof. Said mutant M. mazei PyIRS comprises one or more than one amino acid alteration (independently selected from substitutions, additions and deletions) relative to the corresponding wildtype M. mazei PylRS. According to specific embodiments, such amino acid altera- tions are selected from amino acid substitutions Y306A and Y384F. For example, the PyIRS of the invention comprises mazei PyIRSAF, or an enzymatically active fragment thereof. The amino acid sequence of M. mazei PyIRSAF is set forth in SEQ ID NO:2. SEQ ID NO:2 MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARAL 60 RHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLE 120 NTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMS 180 APVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERE 240 NYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPM 300 LAPNLANYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLE 360 SIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGA 420 GFGLERLLKVKHDFKNIKRAARSESYYNGISTNL 454 According to a further particular embodiment, the PyIRS of the invention comprises M. barkeri wildtype PylRS, or an enzymatically active fragment thereof. The amino acid sequence of wildtype M. barkeri PyIRS is set forth in SEQ ID NO:3. SEQ ID NO:3 MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVNNSRSCRTARAF 60 RHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVSAPKVKKAMPKSVSRAPKPLEN 120 SVSAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRVEALLSPEDKISLNMAKP 180 FRELEPELVTRRKNDFQRLYTNDREDYLGKLERDITKFFVDRGFLEIKSPILIPAEYVER 240 MGINNDTELSKQIFRVDKNLCLRPMLAPTLYNYLRKLDRILPGPIKIFEVGPCYRKESDG 300 KEHLEEFTMVNFCQMGSGCTRENLEALIKEFLDYLEIDFEIVGDSCMVYGDTLDIMHGDL 360 ELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHGFKNIKRASRSESYYNGISTNL 419 The term "nuclear export signal" (abbreviated as "NES") refers to an amino acid se- quence which can direct a polypeptide containing it (such as a NES-containing PyIRS of the invention) to be exported from the nucleus of a eukaryotic cell. Said export is believed to be mostly mediated by Crm1 (chromosomal region maintenance 1, also known as karyopherin exportin 1). NESs are known in the art. For example, the data- base ValidNESs provides sequence information of experimentally validated NES- containing proteins. Further, NES databases like, e.g., NESbase 1.0 (see Le Cour et al., Nucl Acids Res 31(1), 2003) as well as tools for NES prediction like NetNES (see La Cour et al., La Cour et al., Protein Eng Des Sel 17(6):527-536, 2004), NESpredictor (NetNES; see Fu et al., Nucl Acids Res 41:D338-D343, 2013; La Cour et al., Protein Eng Des Sel 17(6):527-536, 2004)) and NESsential (a web interface combined with ValidNESs) are available to the public. Hydrophobic leucine-rich NESs are most com- mon and represent the best characterized group of NESs to date. A hydrophobic leu- cine-rich NES is a non-conservative motif having 3 or 4 hydrophobic residues. Many of these NESs comprise the conserved amino acid sequence pattern LxxLxL (SEQ ID NO:4) or LxxxLxL (SEQ ID NO:5), wherein each L is independently selected from leu- cine, isoleucine, valine, phenylalanine and methionine amino acid residues, and each x is independently selected from any amino acid (see La Cour et al., Protein Eng Des Sel 17(6):527-536, 2004). The term "nuclear localization signal" (abbreviated as "NLS", also referred to in the art as "nuclear localization sequence") refers to an amino acid sequence which can direct a polypeptide containing it (e.g., a wild-type archaeal PyIRS) to be imported into the nucleus of a eukaryotic cell. Said export is believed to be mediated by binding of the NLS-containing polypeptide to importin (also known as karyopherin) so as to form a complex that moves through a nuclear pore. NLSs are known in the art. A multitude of NLS databases and tools for NLS prediction are available to the public, such as NLSdb (see Nair et al., Nucl Acids Res 31(1), 2003), cNLS Mapper (see Kosugi et al., Proc Natl Acad Sci U S A. 106(25):10171-10176, 2009; Kosugi et al., J Biol Chem 284(1):478-485, 2009), SeqNLS (see Lin et al., PLoS One 8(10):e76864, 2013), and NucPred (see Branmeier et al., Bioinformatics 23(9):1159-60, 2007). Archaeal PyIRSs of the invention can be prepared by modifying the amino acid se- quence of a naturally occurring archaeal PyIRS, in particular by introducing one or more amino acid alteration (independently selected from amino acid substitutions, dele- tions and additions) which removes the NLS found in said naturally occurring PyIRS and / or introduces at least one NES. The NLS in the naturally occurring PyIRS can be identified using known NLS detection tools such as, e.g., cNLS Mapper. The removal of a NLS from and / or the introduction of a NES into a polypeptide, such as an archaeal PylRS, can change the localization of the thus modified polypeptide when expressed in a eukaryotic cell, and in particular can avoid or reduce accumulation of the polypeptide in the nucleus of the eukaryotic cell. Thus, the localization of a PyIRS of the invention expressed in a eukaryotic cell can be changed compared to a PylRS, which differs from the PyIRS of the invention in that it (still) comprises the NLS and lacks the NES. Where the archaeal PyIRS of the invention comprises a NES but (still) comprises an NLS, the NES is preferably chosen such that the strength of the NES overrides the NLS preventing an accumulation of the PyIRS in the nucleus of a eukaryotic cell. Removal of the NLS from a wild-type or mutant PyIRS and / or introduction of a NES into the wild-type or mutant PyIRS so as to obtain a PyIRS of the invention do not abrogate PyIRS enzymatic activity. Preferably, PyIRS enzymatic activity is maintained at basical- ly the same level, i.e. the PylRS of the invention has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the enzymatic activity of the corre- sponding wild-type or mutant PyIRS. The NES is expediently located within the PyIRS of the invention such that the NES is functional. For example, a NES can be attached to the C-terminus (e.g., C-terminal of the last amino acid residue) or the N-terminus (e.g., in between amino acid residue 1, the N-terminal methionine, and amino acid residue 2) of a wild-type or mutant archaeal PyIRS. NES sequences suitable in PyIRS of the invention are known in the art (e.g., from NES databases). In one embodiment, the PyIRS of the invention comprises a hydrophobic leucine-rich NES, in particular a NES comprising the amino acid sequence LxxLxL (SEQ ID NO:4) or LxxxLxL (SEQ ID NO:5), wherein each L is independently selected from leucine, isoleucine, valine, phenylalanine and methionine, and each x is inde- pendently selected from any amino acid; more particularly a NES comprising an amino acid sequence selected from L1xxL2xxL1xL3 (SEQ ID NO:6), L1xxxL2xxL1xL3 (SEQ ID NO:7), L1xxL2xxxL1xL3 (SEQ ID NO:8) and L1xxxL2xxxL1xL3 (SEQ ID NO:9), wherein L1 is leucine, L2 is selected from leucine, isoleucine, valine, phenylalanine and methio- nine, L3 is selected from leucine and isoleucine, and each x is independently selected from any amino acid. Preferably, the NES comprises the amino acid sequence LPPLERLTL (SEQ ID NO:10) which is found in the HIV-1 Rev protein or, more prefera- bly, the amino acid sequence ACPVPLQLPPLERLTLD (SEQ ID NO:11). According to a particular embodiment, the PyIRS of the invention comprises an enzy- matically active fragment of the amino acid sequence of M. mazei PyIRSAF set forth in SEQ ID NO:2, and a NES comprising the amino acid sequence of SEQ ID NO:10 or 11. A preferred example of such PyIRS comprises or essentially consists of the amino acid sequence of SEQ ID NO:12. SEQ ID NO:12 MACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACG 60 DHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTR 120 TKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGA 180 TASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESEL 240 LSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTE 300 LSKQIFRVDKNFCLRPMLAPNLANYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFT 360 MLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVG 420 PIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL 471 (underlined: SEQ ID NO:10, bold: SEQ ID NO:11) A further preferred example of such PyIRS comprises or essentially consists of the amino acid sequence of SEQ ID NO:13. SEQ ID NO:13 MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARAL 60 RHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLE 120 NTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMS 180 APVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERE 240 NYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPM 300 LAPNLANYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLE 360 SIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGA 420 GFGLERLLKVKHDFKNIKRAARSESYYNGISTNLACPVPLQLPPLERLTLD 471 (underlined: SEQ ID NO:10, bold: SEQ ID NO:11) According to a particular embodiment, the PyIRS of the invention comprises an enzy- matically active fragment of the amino acid sequence of wildtype M. mazei PyIRS set forth in SEQ ID NO:1, and a NES comprising the amino acid sequence of SEQ ID NO:10 or 11. A preferred example of such PyIRS comprises or essentially consists of the amino acid sequence of SEQ ID NO:14. SEQ ID NO:14 MACPVPLQLPPLERLTLDDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACG 60 DHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTR 120 TKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGA 180 TASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESEL 240 LSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTE 300 LSKQIFRVDKNFCLRPMLAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFT 360 MLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVG 420 PIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL 471 (underlined: SEQ ID NO:10, bold: SEQ ID NO:11) A further preferred example of such PyIRS comprises or essentially consists of the amino acid sequence of SEQ ID NO:15. SEQ ID NO:15 MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARAL 60 RHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLE 120 NTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMS 180 APVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERE 240 NYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPM 300 LAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLE 360 SIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGA 420 GFGLERLLKVKHDFKNIKRAARSESYYNGISTNLACPVPLQLPPLERLTLD 471 (underlined: SEQ ID NO:10, bold: SEQ ID NO:11) According to a further particular embodiment, the PyIRS of the invention comprises an enzymatically active fragment of the amino acid sequence of M. barkeri PyIRS set forth in SEQ ID NO:3, and a NES comprising the amino acid sequence of SEQ ID NO:10 or 11. A preferred example of such PyIRS comprises or essentially consists of the amino acid sequence of SEQ ID NO:16. SEQ ID NO:16 MACPVPLQLPPLERLTLDDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACG 60 DHLVVNNSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVSAPKV 120 KKAMPKSVSRAPKPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRV 180 EALLSPEDKISLNMAKPFRELEPELVTRRKNDFQRLYTNDREDYLGKLERDITKFFVDRG 240 FLEIKSPILIPAEYVERMGINNDTELSKQIFRVDKNLCLRPMLAPTLYNYLRKLDRILPG 300 PIKIFEVGPCYRKESDGKEHLEEFTMVNFCQMGSGCTRENLEALIKEFLDYLEIDFEIVG 360 DSCMVYGDTLDIMHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHGFKNIK 420 RASRSESYYNGISTNL 436 (underlined: SEQ ID NO:10, bold: SEQ ID NO:11) A further preferred example of such PyIRS comprises or essentially consists of the amino acid sequence of SEQ ID NO:17. 13 SEQ ID NO:17 MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVNNSRSCRTARAF 60 RHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVSAPKVKKAMPKSVSRAPKPLEN 120 SVSAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRVEALLSPEDKISLNMAKP 180 FRELEPELVTRRKNDFQRLYTNDREDYLGKLERDITKFFVDRGFLEIKSPILIPAEYVER 240 MGINNDTELSKQIFRVDKNLCLRPMLAPTLYNYLRKLDRILPGPIKIFEVGPCYRKESDG 300 KEHLEEFTMVNFCQMGSGCTRENLEALIKEFLDYLEIDFEIVGDSCMVYGDTLDIMHGDL 360 ELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHGFKNIKRASRSESYYNGISTNLA 420 CPVPLQLPPLERLTLD 436 (underlined: SEQ ID NO:10, bold: SEQ ID NO:11) The PyIRS of the invention are used in tRNAPyI / PyIRS pairs, wherein the PyIRS is ca- pable of acylating the tRNAPyl, preferably with an UAA or a salt thereof. Unless indicated otherwise, "tRNAPyl", as used herein, refers to a tRNA that can be acylated (preferably selectively) by a PyIRS of the invention. The tRNA Pyl described herein in the context of the present invention may be a wildtype tRNA that can be acyl- ated by a PyIRS with pyrrolysine, or a mutant of such tRNA, e.g., a wildtype or a mu- tant tRNA from an archaeon, for example from a Methanosarcina species, e.g. M. mazei or M. barkeri. For site-specific incorporation of the UAA into POI, the anticodon comprised by the tRNAPyl used together with the PyIRS of the invention is expediently the reverse complement of a selector codon. In particular embodiments, the anticodon of the tRNA Pyl is the reverse complement of the amber stop codon. For other applica- tions such as, e.g., proteome labeling (Elliott et al., Nat Biotechnol 32(5):465-472, 2014), the anticodon comprised by the tRNAPyl used together with the PylRS of the invention may be a codon recognized by endogenous tRNAs of the eukaryotic cells. The term "selector codon" as used herein refers to a codon that is recognized (i.e. bound) by the tRNAPyl in the translation process and is not recognized by endogenous tRNAs of the eukaryotic cell. The term is also used for the corresponding codons in polypeptide-encoding sequences of polynucleotides which are not messenger RNAs (mRNAs), e.g. DNA plasmids. Preferably, the selector codon is a codon of low abun- dance in naturally occurring eukaryotic cells. The anticodon of the tRNAPyl binds to a selector codon within an mRNA and thus incorporates the UAA site-specifically into the growing chain of the polypeptide encoded by said mRNA. The known 64 genetic (tri- plet) codons code for 20 amino acids and three stop codons. Because only one stop codon is needed for translational termination, the other two can in principle be used to encode non-proteinogenic amino acids. For example, the amber codon, UAG, has been successfully used as a selector codon in in vitro and in vivo translation systems to direct the incorporation of unnatural amino acids. Selector codons utilized in methods of the present invention expand the genetic codon framework of the protein biosynthet- ic machinery of the translation system used. Specifically, selector codons include, but are not limited to, nonsense codons, such as stop codons, e.g., amber (UAG), ochre (UAA), and opal (UGA) codons; codons consisting of more than three bases (e.g., four base codons); and codons derived from natural or unnatural base pairs. For a given system, a selector codon can also include one of the natural three base codons (i.e. natural triplets), wherein the endogenous translation system does not (or only scarcely) use said natural triplet, e.g., a system that is lacking a tRNA that recognizes the natural triplet or a system wherein the natural triplet is a rare codon. A recombinant tRNA that alters the reading of an mRNA in a given translation system (e.g. a eukaryotic cell) such that it allows for reading through, e.g., a stop codon, a four base codon, or a rare codon, is termed suppressor tRNA. The suppression efficiency for a stop codon serving as a selector codon (e.g., the amber codon) depends upon the competition between the (aminoacylated) tRNAPyl (which acts as suppressor tRNA) and the release factor (e.g. RF1) which binds to the stop codon and initiates release of the growing polypeptide chain from the ribosome. Suppression efficiency of such stop co- don can therefore be increased using a release factor-(e.g. RF1-)deficient strain. A polynucleotide sequence encoding a target polypeptide (also referred to herein as polypeptide of interest or POI) can comprise one or more, e.g., two or more, more than three, etc., codons (e.g. selector codons) which are the reverse complement of the anticodon comprised by the tRNAPyl. Conventional site-directed mutagenesis can be used to introduce said codon(s) at the site of interest into a polynucleotide sequence so as to generate a POI-encoding polynucleotide sequence. A POI comprising one or more than one UAA residue can be prepared according to the present invention using a eukaryotic cell. The eukaryotic cell comprises (e.g., is fed with) at least one unnatural amino acid or a salt thereof corresponding to the UAA resi- due(s) of the POI to be prepared. The eukaryotic cell further comprises: (i) a PyIRS of the invention and a tRNAPyI, wherein the PyIRS is capable of (prefera- bly selectively) acylating the tRNAPyl with the UAA or salt thereof; and (ii) a polynucleotide encoding the POI, wherein any position of the POI occupied by an UAA residue is encoded by a codon (e.g. selector codon) that is the reverse com- plement of the anticodon of the tRNAPyl. The eukaryotic cell is cultured so as to allow translation of the POI-encoding polynucle- otide (ii), thereby producing the POI. For producing a POI (target polypeptide) according to a method of the present inven- tion, the translation in step (b) can be achieved by culturing the eukaryotic cell under suitable conditions, preferably in the presence of (e.g., in a culture medium containing) the UAA or salt thereof, for a time suitable to allow translation at a ribosome of the cell. Depending on the polynucleotide(s) encoding the POI (and optionally the PyIRS, tRNAPyl), it may be required to induce expression by adding a compound inducing tran- scription, such as, e.g., arabinose, isopropyl <semantics>β<annotation encoding="application / x-tex">\beta< / annotation>< / semantics>-D-thiogalactoside (IPTG) or tetracycline. mRNA that encodes the target polypeptide (and comprises one or more than codon that is the reverse complement of the anticodon comprised by the tRNAPyl ) is bound by the ribosome. Then, the polypeptide is formed by stepwise attachment of amino acids and UAAs at positions encoded by codons which are recognized (bound) by respective aminoacyl tRNAs. Thus, the UAA(s) is / are incorporated in the target polypeptide at the position(s) encoded by the codon(s) that is / are the reverse complement of the antico- don comprised by the tRNAPyl. The eukaryotic cell may comprise a polynucleotide sequence encoding the PyIRS of the invention which allows for expression of the PyIRS by the cell. Likewise, the tRNA PyI may be produced by the eukaryotic cell based on a tRNAPyl-encoding polynucleotide sequence comprised by the cell. The PyIRS-encoding polynucleotide sequence and the tRNAPyl-encoding polynucleotide sequence can be located either on the same polynu- cleotide or on separate polynucleotides. Thus, in one embodiment, the present invention provides a method for producing a POI comprising one or more than one UAA residue, wherein the method comprises the steps of: (a) providing a eukaryotic cell comprising polynucleotide sequences encoding: - at least one PyIRS of the invention, - at least one tRNA (tRNA Pyl) that can be acylated by the PylRS, and - at least one POI, wherein any position of the POI occupied by an UAA residue is encoded by a codon that is the reverse complement of the anticodon of the tRNAPyl; and (b) allowing for translation of the polynucleotide sequences by the eukaryotic cell in the presence of an UAA or a salt thereof, thereby producing the PyIRS, tRNAPyI and the POI. The eukaryotic cells used for preparing a POI comprising one or more than one unnat- ural amino acid residue as described herein can be prepared by introducing polynucle- otide sequences encoding the PyIRS, the tRNAPyl and the POI into a eukaryotic (host) cell. Said polynucleotide sequences can be located on the same polynucleotide or on separate polynucleotides, and can be introduced into the cell by methods known in the art (such as, e.g., using virus-mediated gene delivery, electroporation, microinjection, lipofection, or others). The present invention also provides polynucleotides encoding the PyIRS of the inven- tion. In addition to the PyIRS of the invention, such polynucleotide may encode a tRNAPyl that can be acylated by the PylRS. The present invention further provides combinations of at least one polynucleotide en- coding a PyIRS of the invention and at least one polynucleotide encoding a tRNA Pyl that can be acylated by said PyIRS. The polynucleotides of the invention as well as the tRNAPyl- and / or POI-encoding poly- nucleotides used in the context of the present invention are preferably expression vec- tors suitable for transfecting a eukaryotic cell and allowing for the expression of the encoded PyIRS, tRNA Pyl and POI, respectively, in said cell. The present invention also provides a eukaryotic cell capable of expressing a PyIRS of the invention. In particular, the present invention provides a eukaryotic cell comprising a polynucleotide or combination of polynucleotides, wherein said polynucleotide(s) en- code(s) the PyIRS of the invention and a tRNAPyI, wherein the tRNAPyI is a tRNA that can be acylated (preferably selectively) by the PyIRS. Expediently, the eukaryotic cell of the invention is capable of expressing both the tRNAPyl and the PylRS of the inven- tion, wherein the PyIRS is capable of acylating the tRNA pyl (preferably selectively) with an amino acid, e.g. with an UAA. Eukaryotic cells of the present invention can be selected from, but are not limited to, mammalian cells, insect cells, yeast cells and plant cells. The eukaryotic cells of the invention may be present as individual cells or may be part of a tissue (e.g. a cell in a (cultured) tissue, organ or entire organism). The PyIRS and tRNAPyI of the present invention are preferably orthogonal. The term "orthogonal" as used herein refers to a molecule (e.g., an orthogonal tRNA and / or an orthogonal RS) that is used with reduced efficiency by a translation system of interest (e.g., a eukaryotic cell used for expression of a POI as described herein). "Or- thogonal" refers to the inability or reduced efficiency, e.g., less than 20% efficient, less than 10% efficient, less than 5% efficient, or e.g., less than 1% efficient, of an orthogo- nal tRNA or an orthogonal RS to function with the endogenous RSs or endogenous tRNAs, respectively, of the translation system of interest. Accordingly, in particular embodiments of the invention, any endogenous RS of the eukaryotic cell of the invention catalyzes acylation of the (orthogonal) tRNAPyl with re- duced or even zero efficiency, when compared to acylation of an endogenous tRNA by the endogenous RS, for example less than 20% as efficient, less than 10% as efficient, less than 5% as efficient or less than 1% as efficient. Alternatively or additionally, the (orthogonal) PyIRS of the invention acylates any endogenous tRNA of the eukaryotic cell of the invention with reduced or even zero efficiency, as compared to acylation of the tRNAPyl by an endogenous RS of the cell, for example less than 20% as efficient, less than 10% as efficient, less than 5% as efficient or less than 1% as efficient. Unless indicated differently, the terms "endogenous tRNA" and "endogenous aminoacyl tRNA synthetase" ("endogenous RS") used therein refer to a tRNA and an RS, respec- tively, that was present in the cell ultimately used as translation system prior to intro- ducing the PyIRS of the invention and the tRNAPyI, respectively, used in the context of the present invention. The term "translation system" generally refers to a set of components necessary to incorporate a naturally occurring amino acid in a growing polypeptide chain (protein). Components of a translation system can include, e.g., ribosomes, tRNAs, aminoacyl tRNA synthetases (RS), mRNA and the like. Translation systems include artificial mix- ture of said components, cell extracts and living cells, e.g. living eukaryotic cells. The pair of PyIRS and tRNA Pyl used for preparing a POI according to the present inven- tion is preferably orthogonal in that the tRNAPyl, in the eukaryotic cell used for preparing the POI, is preferentially acylated by the PyIRS of the invention with an UAA or a salt thereof (UAA). Expediently, the orthogonal pair functions in said eukaryotic cell such that the cell uses the UAA-acylated tRNAPyl to incorporate the UAA residue into the growing polypeptide chain of the POI. Incorporation occurs in a site-specific manner, e.g., the tRNAPyl recognizes a codon (e.g., a selector codon such as an amber stop codon) in the mRNA coding for the POI. As used herein, the term "preferentially acylated" refers to an efficiency of, e.g., about 50% efficient, about 70% efficient, about 75% efficient, about 85% efficient, about 90% efficient, about 95% efficient, or about 99% or more efficient, at which the PyIRS acyl- ates the tRNAPyl with an UAA compared to an endogenous tRNA or amino acid of a eukaryotic cell. The UAA is then incorporated into a growing polypeptide chain with high fidelity, e.g., at greater than about 75%, greater than about 80%, greater than about 90%, greater than about 95%, or greater than about 99% or more efficiency for a given codon (e.g., selector codon) that is the reverse complement of the anticodon comprised by the tRNAPyl. tRNAPyl / PyIRS pairs suitable in producing a POI according to the present invention may be selected from libraries of mutant tRNA and PylRSs, e.g. based on the results of a library screening. Such selection may be performed analogous to known methods for evolving tRNA / RS pairs described in, e.g., WO 02 / 085923 and WO 02 / 06075. To gen- erate a tRNAPyl / PylRS pair of the invention, one may start from a wild-type or mutant archaeal PyIRS that (still) comprises a nuclear localization signal and lacks a NES, and remove the nuclear localization signal and / or introduce a NES prior to or after a suita- ble tRNAPyl / PylRS pair is identified. After translation, the target polypeptide prepared according to the present invention may optionally be recovered and purified, either partially or substantially to homogenei- ty, according to procedures generally known in the art. Unless the target polypeptide is secreted into the culture medium, recovery usually requires cell disruption. Methods of cell disruption are well known in the art and include physical disruption, e.g., by (ultra- sound) sonication, liquid-sheer disruption (e.g., via French press), mechanical methods (such as those utilizing blenders or grinders) or freeze-thaw cycling, as well as chemi- cal lysis using agents which disrupt lipid-lipid, protein-protein and / or protein-lipid inter- actions (such as detergents), and combinations of physical disruption techniques and chemical lysis. Standard procedures for purifying polypeptides from cell lysates or cul- ture media are also well known in the art and include, e.g., ammonium sulfate or etha- nol precipitation, acid or base extraction, column chromatography, affinity column chromatography, anion or cation exchange chromatography, phosphocellulose chro- matography, hydrophobic interaction chromatography, hydroxylapatite chromatog- raphy, lectin chromatography, gel electrophoresis and the like. Protein refolding steps can be used, as desired, in making correctly folded mature proteins. High performance liquid chromatography (HPLC), affinity chromatography or other suitable methods can be employed in final purification steps where high purity is desired. Antibodies made against the polypeptides of the invention can be used as purification reagents, i.e. for affinity-based purification of the polypeptides. A variety of purification / protein folding methods are well known in the art, including, e.g., those set forth in Scopes, Protein Purification, Springer, Berlin (1993); and Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press (1990); and the references cited therein. As noted, those of skill in the art will recognize that, after synthesis, expression and / or purification, polypeptides can possess a conformation different from the desired con- formations of the relevant polypeptides. For example, polypeptides produced by pro- karyotic systems often are optimized by exposure to chaotropic agents to achieve proper folding. During purification from, e.g., lysates derived from E. coli, the expressed polypeptide is optionally denatured and then renatured. This is accomplished, e.g., by solubilizing the proteins in a chaotropic agent such as guanidine HCl. In general, it is 19 occasionally desirable to denature and reduce expressed polypeptides and then to cause the polypeptides to re-fold into the preferred conformation. For example, guani- dine, urea, DTT, DTE, and / or a chaperonin can be added to a translation product of interest. Methods of reducing, denaturing and renaturing proteins are well known to those of skill in the art. Polypeptides can be refolded in a redox buffer containing, e.g., oxidized glutathione and L-arginine. The term "unnatural amino acid" (abbreviated "UAA"), as used herein, refers to an ami- no acid that is not one of the 20 canonical amino acids or selenocysteine or pyrroly- sine. The term also refers to amino acid analogs, e.g. compounds which differ from amino acids such that the <semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>-amino group is replaced by a hydroxyl group and / or the carboxylic acid function forms an ester. When translationally incorporated into a poly- peptide, said amino acid analogs yield amino acid residues which are different from the amino acid residues corresponding to the 20 canonical amino acids or selenocysteine or pyrrolysine. When UAAs which are amino acid analogs wherein the carboxylic acid function forms an ester of formula -C(O)-O-R are used for preparing polypeptides in a translation system (such as a eukaryotic cell), it is believed that R is removed in situ, for example enzymatically, in the translation system prior of being incorporated in the POI. Accordingly, R is expediently chosen so as to be compatible with the translation system's ability to convert the UAA or salt thereof into a form that is recognized and processed by the PyIRS of the invention. UAAs useful in methods and kits of the present invention have been described in the prior art (for review see e.g. Liu et al., Annu Rev Biochem 83:379-408, 2010, Lemke, ChemBioChem 15:1691-1694, 2014). The UAAs may comprise a group (herein referred to as "labeling group") that facilitates reaction with a suitable group (herein referred to as "docking group") of another mole- cule (herein termed "conjugation partner molecule") so as to covalently attach the con- jugation partner molecule to the UAA. When a UAA comprising a labeling group is translationally incorporated into a target polypeptide, the labeling group becomes part of the target polypeptide. Accordingly, a target polypeptide prepared according to the method of the present invention can be reacted with one or more than one conjugation partner molecule such that the conjugation partner molecules bind covalently to the (labeling groups of the) unnatural amino acid residue(s) of the target polypeptide. Such conjugation reactions may be used for in situ coupling of target polypeptides within a cell or tissue expressing the target polypeptide, or for site-specific conjugation of isolat- ed or partially isolated target polypeptides. Particular useful choices for combinations of labeling groups and docking groups (of conjugation partner molecules) are those which can react by metal-free click reactions. Such click reactions include strain-promoted inverse-electron-demand Diels-Alder cy- cloadditions (SPIEDAC; see, e.g., Devaraj et al., Angew Chem Int Ed Engl 2009, 48:7013)) as well as cycloadditions between strained cycloalkynyl groups, or strained cycloalkynyl analog groups having one or more of the ring atoms not bound by the tri- ple bond substituted by amino groups), with azides, nitrile oxides, nitrones and diazo- carbonyl reagents (see, e.g., Sanders et al., J Am Chem Soc 2010, 133:949; Agard et al., J Am Chem Soc 2004, 126:15046), for example strain promoted alkyne-azide cy- cloadditions (SPAAC). Such click reactions allow for ultrafast and biorthogonal covalent site-specific coupling of UAA labeling groups of target polypeptides with suitable groups of coupling partner molecule. Pairs of docking and labeling groups which can react via the above-mentioned click reactions are known in the art. Examples of suitable UAAs comprising docking groups include, but are not limited to, the UAAs described, e.g., in WO 2012 / 104422 and WO 2015 / 107064. Examples of particular suitable pairs of docking groups (comprised by the conjugation partner molecule) and labeling groups (comprised by the UAA residue(s) of the POI) include but are not limited to: (a) a docking group comprising (or essentially consisting of) a group selected from an azido group, a nitrile oxide functional group (i.e. a radical of formula, a nitrone func- tional group or a diazocarbonyl group, combined with a labeling group comprising (or essentially consisting of) an optionally substituted strained alkynyl group (such groups can react covalently in a copper-free strain promoted alkyne-azide cycload- dition (SPAAC)); (b) a docking group comprising (or essentially consisting of) an optionally substituted strained alkynyl group, combined with a labeling group comprising (or essentially consisting of) a group selected from an azido group, a nitrile oxide functional group (i.e. a radical of formula, a nitrone functional group or a diazocarbonyl group (such groups can react covalently in a copper-free strain promoted alkyne-azide cycload- dition (SPAAC)); (c) a docking group comprising (or essentially consisting of) a group selected from op- tionally substituted strained alkynyl groups, optionally substituted strained alkenyl groups and norbornenyl groups, combined with a labeling group comprising (or es- sentially consisting of) an optionally substituted tetrazinyl group (such groups can react covalently in a copper-free strain promoted inverse-electron-demand Diels- Alder cycloaddition (SPIEDAC)). (d) a docking group comprising (or essentially consisting of) an optionally substituted tetrazinyl group, combined with a labeling group comprising (or essentially consist- ing of) a group selected from optionally substituted strained alkynyl groups, option- ally substituted strained alkenyl groups and norbornenyl groups (such groups can react covalently in a copper-free strain promoted inverse-electron-demand Diels- Alder cycloaddition (SPIEDAC)). Optionally substituted strained alkynyl groups include, but are not limited to, optionally substituted trans-cyclooctenyl groups, such as those described in. Optionally substitut- ed strained alkenyl groups include, but are not limited to, optionally substituted cy- clooctynyl groups, such as those described in WO 2012 / 104422 and WO 2015 / 107064. Optionally substituted tetrazinyl groups include, but are not limited to, those described in WO 2012 / 104422 and WO 2015 / 107064 An azido group is a radical of formula -N3. A nitrone functional group is a radical of formula -C(Rx)=N+(Ry)-O-, wherein Rx and Ry are organic residues, e.g., residues independently selected from C1-C6-alkyl as de- scribed herein. A diazocarbonyl group is a radical of formula <semantics>−C(O)−CH=N2<annotation encoding="application / x-tex">-C(O)-CH=N_2< / annotation>< / semantics>. A nitrile oxide functional group is a radical of formula -C≡N+-O- or, preferably, of formu- Ia −C=N+(Rx)-O-, wherein Rx is an organic residue, e.g., a residue selected from C1-C6- alkyl as described herein. "Cyclooctynyl is an unsaturated cycloaliphatic radical having 8 carbon atoms and one triple bond in the ring structure. "Trans-cyclooctenyl" is an unsaturated cycloaliphatic radical having 8 carbon atoms and one double bond that is in trans configuration in the ring structure. "Tetrazinyl" is a 6-membered monocyclic aromatic radical having 4 nitrogen ring atoms and 2 carbon ring atoms. Unless indicated otherwise, the term "substituted" means that a radical is substituted with 1, 2 or 3, especially 1 or 2, substituent(s). In particular embodiments, these sub- stituents can be selected independently from hydrogen, halogen, C1-C4-alkyl, <semantics>(RaO)2P(O)O−C1−C4−alkyl,(RbO)2P(O)−C1−C4−alkyl,CF3,CN,hydroxyl,C1−C4−alkoxy,−O−<annotation encoding="application / x-tex">(R^aO)_2P(O)O-C_1-C_4-alkyl, (R^bO)_2P(O)-C_1-C_4-alkyl, CF_3, CN, hydroxyl, C_1-C_4-alkoxy, -O-< / annotation>< / semantics> CF3, C2-C5-alkenoxy, C2-C5-alkanoyloxy, C1-C4-alkylaminocarbonyloxy or C1-C4- alkylthio, C1-C4-alkylamino, di-(C1-C4-alkyl)amino, C2-C5-alkenylamino, N-C2-C5- alkenyl-N-C1-C4-alkyl-amino and di-(C2-C5-alkenyl)amino, wherein Ra and Rb Ra, Rb are independently hydrogen or C2-C5-alkanoyloxymethyl. The term halogen denotes in each case a fluorine, bromine, chlorine or iodine radical, in particular a fluorine radical. C1-C4-Alkyl is a straight-chain or branched alkyl group having from 1 to 4, in particular from 1 to 3 carbon atoms. Examples include methyl and C2-C4-alkyl such as ethyl, n- propyl, iso-propyl, n-butyl, 2-butyl, iso-butyl and tert-butyl. C2-C5-Alkenyl is a singly unsaturated hydrocarbon radical having 2, 3, 4 or 5 carbon atoms. Examples include vinyl, allyl (2-propen-1-yl), 1-propen-1-yl, 2-propen-2-yl, me- thallyl (2-methylprop-2-en-1-yl), 1-methylprop-2-en-1-yl, 2-buten-1-yl, 3-buten-1-yl, 2- penten-1-yl, 3-penten-1-yl, 4-penten-1-yl, 1-methylbut-2-en-1-yl and 2-ethylprop-2-en- 1-yl. C1-C4-Alkoxy is a radical of formula R-O-, wherein R is a C1-C4-alkyl group as defined herein. C2-C5-Alkenoxy is a radical of formula R-O-, wherein R is C2-C5-alkenyl as defined herein. C2-C5-Alkanoyloxy is a radical of formula R-C(O)-O-, wherein R is C1-C4-alkyl as de- fined herein. C1-C4-Alkylaminocarbonyloxy is a radical of formula R-NH-C(O)-O-, wherein R is C1-C4- alkyl as defined herein. <semantics>C1<annotation encoding="application / x-tex">C_1< / annotation>< / semantics>-<semantics>C4<annotation encoding="application / x-tex">C_4< / annotation>< / semantics>-Alkylthio is a radical of formula R-S-, wherein R is <semantics>C1<annotation encoding="application / x-tex">C_1< / annotation>< / semantics>-<semantics>C4<annotation encoding="application / x-tex">C_4< / annotation>< / semantics>-alkyl as defined herein. C1-C4-Alkylamino is a radical of formula R-NH-, wherein R is C1-C4-alkyl as defined herein. Di-(C1-C4-alkyl)amino is a radical of formula Rx-N(Ry)-, wherein Rx and Ry are inde- pendently C1-C4-alkyl as defined herein. C2-C5-Alkenylamino is a radical of formula R-NH-, wherein R is C2-C5-alkenyl as de- fined herein. N-C2-C5-alkenyl-N-C1-C4-alkyl-amino is a radical of formula Rx-N(Ry)-, wherein Rx is C2- C5-alkenyl as defined herein and Ry is C1-C4-alkyl as defined herein. Di-(C2-C5-alkenyl)amino is a radical of formula Rx-N(Ry)-, wherein Rx and Ry are inde- pendently C2-C5-alkenyl as defined herein. C2-C5-Alkanoyloxymethyl is a radical of formula Rx-C(O)-O-CH2-, wherein Rx is C1-C4- alkyl as defined herein. The UAAs used in the context of the present invention can be used in the form of their salt. Salts of an UAA as described herein mean acid or base addition salts, especially addition salts with physiologically tolerated acids or bases. Physiologically tolerated acid addition salts can be formed by treatment of the base form of an UAA with appro- priate organic or inorganic acids. UAAs containing an acidic proton may be converted into their non-toxic metal or amine addition salt forms by treatment with appropriate organic and inorganic bases. The UAAs and salts thereof described in the context of the present invention also comprise the hydrates and solvent addition forms thereof, e.g. hydrates, alcoholates and the like. Physiologically tolerated acids or bases are in particular those which are tolerated by the translation system used for preparation of POI with UAA residues, e.g. are substan- tially non-toxic to living eukaryotic cells. UAAs, and salts thereof, useful in the context of the present the invention can be pre- pared by analogy to methods which are well known in the art and are described, e.g., in the various publications cited herein. The nature of the coupling partner molecule depends on the intended use. For exam- ple, the target polypeptide may be coupled to a molecule suitable for imaging methods or may be functionalized by coupling to a bioactive molecule. For instance, in addition to the docking group, a coupling partner molecule may comprise a group that selected from, but are not limited to, dyes (e.g. fluorescent, luminescent, or phosphorescent dyes, such as dansyl, coumarin, fluorescein, acridine, rhodamine, silicon-rhodamine, BODIPY, or cyanine dyes), molecules able to emit fluorescence upon contact with a reagent, chromophores (e.g., phytochrome, phycobilin, bilirubin, etc.), radiolabels (e.g. radioactive forms of hydrogen, fluorine, carbon, phosphorous, sulphur, or iodine, such as tritium, 18F, 11C, 14C, 32P, 33P, 33S, 35S, 11In, 125I, 123I, 131I, 212B, 90Y or 186Rh), MRI- sensitive spin labels, affinity tags (e.g. biotin, His-tag, Flag-tag, strep-tag, sugars, lipids, sterols, PEG-linkers, benzylguanines, benzylcytosines, or co-factors), polyethylene glycol groups (e.g., a branched PEG, a linear PEG, PEGs of different molecular 24 weights, etc.), photocrosslinkers (such as p-azidoiodoacetanilide), NMR probes, X-ray probes, pH probes, IR probes, resins, solid supports and bioactive compounds (e.g. synthetic drugs). Suitable bioactive compounds include, but are not limited to, cytotoxic compounds (e.g., cancer chemotherapeutic compounds), antiviral compounds, biologi- cal response modifiers (e.g., hormones, chemokines, cytokines, interleukins, etc.), mi- crotubule affecting agents, hormone modulators, and steroidal compounds. Specific examples of useful coupling partner molecules include, but are not limited to, a mem- ber of a receptor / ligand pair; a member of an antibody / antigen pair; a member of a lec- tin / carbohydrate pair; a member of an enzyme / substrate pair; biotin / avidin; bio- tin / streptavidin and digoxin / antidigoxin. The ability of certain (labeling groups of) UAA residues to be coupled covalently in situ to (the docking groups of) conjugation partner molecules, in particular by a click reac- tion as described herein, can be used for detecting a target polypeptide having such UAA residue(s) within a eukaryotic cell or tissue expressing the target polypeptide, and for studying the distribution and fate of the target polypeptides. Specifically, the method of the present invention for preparing a target polypeptide by expression in eukaryotic cells can be combined with super-resolution microscopy (SRM) to detect the target polypeptide within the cell or a tissue of such cells. Several SRM methods are known in the art and can be adapted so as to utilize click chemistry for detecting a target poly- peptide expressed by a eukaryotic cell of the present invention. Specific examples of such SRM methods include DNA-PAINT (DNA point accumulation for imaging in na- noscale topography; described, e.g., by Jungmann et al., Nat Methods 11:313-318, 2014), dSTORM (direct stochastic optical reconstruction microscopy) and STED (stimulated emission depletion) microscopy. The present invention also provides kits comprising a polynucleotide encoding a PyIRS of the present invention or a eukaryotic cell capable of expressing such PylRS. The kit of the invention may further comprise at least one unnatural amino acid, or a salt thereof, which can be used for acylating a tRNA in a reaction catalyzed by the PylRS. The kit of the invention may also comprise a tRNA that can be acylated by the PyIRS (tRNAPyl). Kits of the invention can be used in methods for preparing UAA-residue con- taining target polypeptides or conjugates thereof as described herein. 25 EXAMPLES Methods (A) Synthesis of UAA Compound 2 (TCO*) was prepared as described in WO 2015 / 107064. (B) Cell culture, transfections and feeding with UAAs HEK293T cells (ATCC CRL-3216) and COS-7 cells (ATCC, CRL-1651) were main- tained in Dulbecco's modified Eagle's medium (Life Technologies, 41965-039) supple- mented with 1% penicillin-streptomycin (Sigma, 10,000 U / ml penicillin, 10 mg / ml strep- tomycin, 0.9% NaCl), 2 mM L-glutamine (Sigma), 1 mM sodium pyruvate (Life Tech- nologies) and 10% FBS (Sigma). Cells were cultured at 37°C in a 5% CO2 atmosphere and passaged every 2-3 days up to 15-20 passages. In all cases, cells were seeded 15-20h prior to transfection at a density resulting in 70- 80% confluency at the time of transfection. Chambers for HEK293T experiments were coated with poly-L-lysine (Sigma) as described in Nikic et al. (Nat Protoc 10(5):780- 791, 2015)). Immunolabeling and FISH were performed on 24-well plates with glass bottom (Greiner Bio-One). All transfections were done using the JetPrime reagent (PeqLab) according to the manufacturer's recommendations. Stock and working solutions for all of the used UAAs were prepared as described in Nikic et al. (Nat Protoc 10(5):780-791, 2015). Unless otherwise stated, final UAA con- centration in the cell culture medium was 250 µM. (C) Preparation of anti-PylRS antibody E. coli BL21(DE3)Al cells were transformed with the plasmid pTXB3-6His-TEV-PyIRSAL and the encoded His-tagged fusion of M. mazei PyIRSAF and TEV (His6-TEV-PyIRSAF) was recombinantly expressed in TB medium overnight at 18°C after induction with 0.02% arabinose and 1 mM IPTG. Cells were harvested by centrifugation, resuspend- ed in 4x PBS (pH 8, 1 mM PMSF, 0.2 mM TCEP) and lysed using a high pressure ho- mogenizer. Debris was removed by centrifugation and His6-TEV-PyIRSAF was purified from the clear supernatant by incubation with Ni-NTA magnetic beads for 1 h at 4°C, washing with increasing imidazole concentrations, and elution with 400 mM imidazole in 4x PBS. The protein containing elution fraction was concentrated using a protein filter device (Spin-X UF, Corning, 30 kDa cutoff). The protein was further purified using preparative gel filtration chromatography (Superdex 200, GE Healthcare). The protein containing fractions were concentrated and used for immunization of two rats (Euro- gentec). The resulting polyclonal anti-PyIRS antibody was used for detecting PyIRSAF and variants therefore in the examples described herein. (D) Flow cytometry Unless stated otherwise, cells were harvested two days after transfection, resuspended in 1x PBS and passed through 70 µm cell strainers. Co-transfections for flow cytometry were performed with a plasmid encoding the POI (including a TAG codon encoding the amino acid position to be occupied by the UAA), a plasmid encoding the tRNAPyl having the anticodon CUA (hereinafter simply referred to as tRNAPyl) and a plasmid encoding the PyIRS or variant thereof, respectively, at a 1:1:1 ratio with 1.2 µg total DNA. Cell culture medium was exchanged for fresh medium containing the UAA 4-6h post- transfection and left until the time of harvesting. Data acquisition and analysis were performed using a LSRFortessa SORP Cell Analyzer (Becton, Dickinson and Compa- ny) and the FlowJo software (FlowJo). Cells were gated first by cell type (using FSC-A x SSC-A parameters) and then by single cell (FSC-A x SSC-W). GFP fluorescence was acquired in the 488-530 / 30 channel and iRFP fluorescence in the 640-730 / 45 channel. (E) PyIRS immunostaining and imaging, fluorescence in situ hybridization (FISH) One day after transfection, the cells were fixed in 2% paraformaldehyde in 1x PBS for 10 min at RT, and then permeabilized in 0.5% Triton in 1x PBS for 15 min at RT. The permeabilized cell samples were incubated for 90 min in blocking solution (3% BSA in 1x PBS for 90 min at RT), and then with the primary antibody (polyclonal anti-PylRS, prepared as described herein, 1 µg / ml in blocking solution) overnight at 4°C. The next day, the cell samples were washed with 1x PBS and incubated with secondary anti- body (Thermo Fisher Scientific, goat anti-rat IgG(H+L) Alexa Fluor 594 conjugate, 2 µg / ml in blocking solution) for 60 min at RT. DNA was stained with Hoechst 33342 (1 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>g / ml in 1xPBS) for 10 min at RT. Fluorescence in situ hybridization (FISH) experiments were performed one day after transfection. The hybridization protocol was adapted for 24-well plates from Pierce et al., Methods Cell Biol 122:415-436, 2014). The hybridization probe (5'- CTAACCCGGCTGAACGGATTTAGAGTCCATTCGATC-3', labelled at the 5' terminus with digoxigenin; SEQ ID NO:18) was used at 0.16 μM. After the washes with SSC, cells were incubated for 1h at RT in blocking buffer (0.1 M TrisHCl, 150 mM NaCl, 1x blocking reagent (Sigma 000000011096176001). Then, cells were incubated with an anti-digoxigenin-fluorescein antibody conjugate (Sigma 00000011207741910) at a 1:200 dilution in blocking buffer overnight at 4°C. The next day, 3 washes of 5 minutes were done in Tween buffer (0.1 M TrisHCl, 150 mM NaCl, 0.5% Tween20). Finally, DNA was stained with Hoechst 33342 (1 µg / ml in 1xPBS) for 10 min at RT. Confocal images were acquired on a Leica SP8 STED 3X microscope using the 405 nm (for Hoechst 33342) and 594 nm (for Alexa594) laser lines for excitation. Emis- sion light was collected with HyD detectors at 420-500 nm and 605-680 nm respective- ly. (F) Vimentin and Nup153: constructs and transfections Specific mutations were introduced into the plasmid DNA sequence of constructs of interest by PCR-based site-directed mutagenesis, thus generating in-frame amber co- dons in the cDNA. For vimentin, the pVimentin-PSmOrange plasmid (Addgene plasmid #31922; Subach et al., Nat Methods 8:771-777, 2011) was mutated at position N116 of vimentin, thus generating the pVimentinN116TAG-PSmOrange construct. For Nup153, a pGFP-Nup153 plasmid was constructed by inserting a codon-optimized Nup153 cDNA into a pEGFP backbone. Subsequently, position N149 of the GFP gene was mutated, thus generating the pGFPN149TAG-Nup153 construct. For the expression of the amber suppression system in mammalian cells, the cells were transfected with the pcDNA3.1 tRNA Pyl / NES-PylRS Plasmid. For Click-PAINT experiments, cells were co-transfected with pcDNA3.1 tRNAPy / NES- PyIRSAF and either pVimentinN116TAG-PSmOrange or pGFPN149TAG-Nup153 and at ratio of a 1:1 using method (B) described herein. UAA 2 (TCO*) was added immediately after transfections. 8-10 hours after transfection, the cell culture medium was ex- changed and cells were cultured overnight with fresh UAA solution. Approximately 30- 36 h after transfection, the cell culture medium was exchanged for fresh medium and cells were cultured overnight without UAA. (G) Click-PAINT labeling Approximately 48 h after transfection, the cells were rinsed with PBS, fixed in 2% para- formaldehyde in 1x PBS for 10 min at RT, and then permeabilized in 0.1 % Triton in 1x PBS for 15 min at RT. The permeabilized cell samples were rinsed with PBS again, prior to labeling. For Click-PAINT labeling, the cells were incubated in 15µM of the docking strand oligonucleotide (5'-ttatacatcta-3', functionalized at the 5' terminus with 1,2,4,5-tetrazine; SEQ ID NO:19) in 1x PBS for 10 min at 37°C, and then rinsed with 1x 28 PBS. Prior to imaging and either on the same day or up to 3 days after cell incubation with the docking strand, the imager strand (5'-ctagatgtat-3', functionalized at the 3' end with Atto655; SEQ ID NO:20) was added to the cells at a final concentration of 800 pM (in 1x PBS, 500 mM NaCl, pH 8, as described in Jungmann et al., Nat Methods 11:313- 318, 2014). (H) Click-PAINT imaging and image processing Click-PAINT microscopy was performed using a Leica GSD microscope, equipped with a Leica HCX PL APO 160x / NA 1.43 oil CORR TIRF PIFOC objective and GFP, Cy3 and Cy5 filter sets. All images were acquired in the TIRF mode. For vimentin imaging, the Cy3 channel (532 nm excitation) was used to identify transfected cells based on the vimentin–mOrange fusion. Due to the position of mOrange at the C terminus only the cells which successfully incorporated the UAA when expressing vimentin–mOrange contributed to the fluorescence signal. For Nup153, a GFP fusion was used to identify transfected cells. Atto655 was excited with a 642 nm laser and images were acquired with 100 ms exposure in the TIRF mode. For each image, 30,000–100,000 frames were acquired. Super-resolution Click-PAINT images were reconstructed using the Localizer Package (Dedecker et al., J Biomed Opt 17:126008, 2012) for IgorPro (Wavemetrics, Portland, USA). Firstly, a threshold based on the maximum likelihood ratio was applied, followed by fitting with a symmetrical 2D Gaussian function for localization of the spots. Sporad- ic long-lasting associations of docking and imager strands were observed, giving rise to repetitive localization in sequential frames. In order to correct for this, identical emitters (falling within one standard deviation of the spot fit) were consolidated into a single intensity-weighed localization. Finally, a super-resolution image was reconstructed from binning all the detected events and convolving the resulting image with a Gaussian width according to the resolution determined by the Fourier ring correlation 2<semantics>σ<annotation encoding="application / x-tex">\sigma< / annotation>< / semantics> criterion for Nup153 and 0.143 criterion for vimentin (Banterle et al., J Struct Biol 183:363-367, 2013). (1) Baculovirus-based transfection of insect cells Following standard protocols, insect cells of line Sf21 were cultured in a protein-free, serum-free standard culture medium for Spodoptera frugiperda cells (Sf-900™ III SFM) at 27°C shaking at 180 rpm. The Sf21 cells were split every day to a density of 0.6 x 106 cells / ml or every third day to a density of 0.3 x 106 cells / ml. Baculovirus shuttle vector (Bacmid) DNA containing an expression cassette encoding tRNAPyl, mCherry-GFPY39TAG and either PyIRSAF or NES-PyIRSAF was prepared using standard cloning and recombination procedures. For transfection 3 ml / well of 0.3 x 106 Sf21 cells / ml were seeded in a 6-well cell culture multidish (Nunclon Delta Surface, Thermo scientific) and transfected with Bacmid DNA using a nonliposomal transfection reagent (FuGENE®HD Transfection Reagent, Promega) following the manufacturer's instructions. V0-virus was harvested 70 h post transfection and the V1-generation was started. Therefor, 25 ml Sf21 cells at 0.6 x 106 cells / ml were transfected with 3 ml of the <semantics>V0<annotation encoding="application / x-tex">V_0< / annotation>< / semantics>-virus. After cell proliferation stopped, the cultures were kept for another 48-60 h at 27°C shaking at 180 rpm. The transfected cells were harvested by centrifugation (500 rpm, 10 min) and the supernatant (i.e., the V1-Virus) was stored at 4°C. (J) Expression experiments using transfected insect cells 5-25 ml Sf21 cells at 0.6 x 106 cells / ml were transduced with V1-virus prepared by method (I) at a ratio of 100:1 vol / vol (cells:virus). One day afterwards, different amounts of UAA 1 (0-1 mM final concentration) was added to the cultures. After three days of culture, the cells were harvested by centrifugation (500 rpm, 10 min), cooled down to 4°C, resuspended in 2 ml sterile 1x PBS, filtered through a cell strainer (Falcon, 70 μm, Fisher Scientific) and kept on ice until analysis. Data for 500,000 cells of each sample was acquired and analyzed using a LSRFortessa SORP Cell Analyzer (Becton, Dickin- son and Company) and the FlowJo software (FlowJo Enterprise). Cells were gated first by cell type (using FSC-A x SSC-A parameters) and then by single cell (FSC-A x SSC- W). GFP fluorescence was acquired in the 488-530 / 30 channel and mCherry fluores- cence in the 561-610 / 20 channel. Example 1: Identification of putative NLS in M. mazei and M. barkeri PyIRS Computational analysis of the amino acid sequences of M. mazei PyIRS and M. barkeri PyIRS (shown below) predicted a putative nuclear localization sequence (NLS, under- lined portion of the PyIRS sequences shown below). SEQ ID NO:1 MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARAL 60 RHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLE 120 NTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMS 180 APVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERE 240 NYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPM 300 30 LAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLE 360 SIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGA 420 GFGLERLLKVKHDFKNIKRAARSESYYNGISTNL 454 SEQ ID NO:3 MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVNNSRSCRTARAF 60 RHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVSAPKVKKAMPKSVSRAPKPLEN 120 SVSAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRVEALLSPEDKISLNMAKP 180 FRELEPELVTRRKNDFQRLYTNDREDYLGKLERDITKFFVDRGFLEIKSPILIPAEYVER 240 MGINNDTELSKQIFRVDKNLCLRPMLAPTLYNYLRKLDRILPGPIKIFEVGPCYRKESDG 300 KEHLEEFTMVNFCQMGSGCTRENLEALIKEFLDYLEIDFEIVGDSCMVYGDTLDIMHGDL 360 ELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHGFKNIKRASRSESYYNGISTNL 419 The NLS motifs were predicted using "cNLS Mapper" (Kosugi et al., Proc Natl Acad Sci USA 106:10171-10176, 2009). Example 2: Intracellular localization of M. mazei PylRSAF In examples 2-5, M. mazei PyIRSAF was used (therein referred to simply as "PyIRSAF"). HEK293T cells and COS-7 cells were transfected with a plasmid facilitating eukaryotic expression of tRNAPyl / PyIRSAF (pcDNA3.1 tRNAPyl / PyIRSAF) and immunostained with polyclonal anti-PyIRS antibody or tRNAPyl was detected by FISH using methods (B) and (E) described herein. As shown in Figures 1a, 2a, 3a and 4a, for both HEK293T and COS-7 cells, strong nuclear and putatively nucleolar immunostaining and FISH signals were detected, while there was almost no signal in the cytoplasm. This indicates that PyIRSAF and tRNAPyI were localized predominantly in the nucleus rather than in the cytoplasm, where trans- lation takes place. Example 3: Amber suppression by PyIRSAF and NES-PyIRSAF A PyIRSAF variant was prepared which differs from PyIRSAF (amino acid sequence set forth in SEQ ID NO:2) in having a strong N-terminal NES ("NES-PyIRSAF", amino acid sequence set forth in SEQ ID NO:12). HEK293T cells were co-transfected with polynucleotides encoding tRNAPyl, iRFP- GFPY39TAG and either PyIRSAF or NES-PyIRSAF using method (B) described herein. HEK293T cells expressing iRFP-GFPY39TAG in the presence or absence of 1 (BOC) were analyzed for GFP and iRFP fluorescence using flow cytometry method (D) de- scribed herein 2 days post-transfection. iRFP-GFPY39TAG is a fusion of iRFP (infrared fluorescent protein) and GFP (green fluo- rescent protein), wherein the permissive site 39 of GFP is encoded by the amber (TAG) stop codon. iRFP-GFP serves as an amber suppression reporter because the cells turn red (express iRFP) if properly transfected but GFP is only produced if the amber codon is suppressed to encode the UAA (here: BOC). The ratio of green fluo- rescence (GFP) to red fluorescence (iRFP) in the cells is therefore an indicator of am- ber suppression efficiency. As shown in Figure 5, NES-PyIRSAF showed significant enhancement of amber sup- pression efficiency (68.6%) compared to PyIRSAF. The differences in efficiency drop were especially noticeable by inspecting the "Bright DPs" (double positive) population. Additional experiments were performed where HEK293T cells were co-transfected with varying amounts of iRFP-GFP (ranging from 100 to 500 ng plasmid per well) and either tRNAPyl / PylRSAF or tRNAPyl / NES-PylRSAF using method (B) described herein, and low (50 µM) or high (250 µM) concentrations of UAA (BOC) were used. Flow cy- tometric analysis of these cells using method (D) described herein confirmed that am- ber suppression efficiency of variant NES-PyIRSAF was significantly enhanced com- pared to PyIRSAF (see Figures 6 and 7). The number of bright GFP-fluorescent cells (i.e., successful iRFP-GFPY39TAG amber suppression) observed cell samples transfect- ed with NES-PyIRSAF and cultured in the presence of 1 was enhanced up to 15-fold compared to the corresponding cell sample transfected with PyIRSAF and cultured in the presence of 1. Example 4: Intracellular localization of NES-PyIRSAF HEK293T cells and COS-7 cells were transfected with a plasmid facilitating eukaryotic expression of tRNAPyl / NES-PylRSAF and immunostained with polyclonal anti-PylRS antibody or tRNAPyl was detected by FISH using methods (B) and (E) described herein. As shown in Figures 1b, 2b, 3b and 4b, for both HEK293T and COS-7 cells, clear cyto- solic immunostaining and FISH signals were detected, while the strong fluorescence in the nucleus observed with PyIRSAF (cf. example 2) was absent. This indicates a cyto- solic distribution of NES-PyIRSAF and tRNAPyI. Example 5: Use of NES-PyIRSAF in super-resolution microscopy The new tRNAPyl / NES-PylRSAF amber suppression pair was used for examining distri- bution of a target polypeptide within transfected HEK293T cells by in super-resolution microscopy using a method termed Click-PAINT that uses elements of the DNA-PAINT microscopy methods described, e.g., by Jungmann et al. (Nat Methods 11:313-318, 2014) The principle of Click-PAINT is outlined in Figure 8a. The cells express a target poly- peptide (POI) comprising an UAA residue. The UAA residue comprises a labeling group (e.g., a trans-cyclooctenyl group). The cell is contacted with a docking strand oligonucleotide carrying a docking group (e.g., a 1,2,4,5-tetrazine group) that reacts via Click reaction (such as SPAAC or SPIEDAC) with the labeling group of the UAA resi- due, thus coupling the docking strand to the POI. Then, an imager strand carrying an imaging group (for example a dye such as Atto655) is added to the cells. Proper choice of the location of the docking group within the docking strand oligonucleotide (e.g., at the 5' end) and the location of the imaging group within the imager strand (e.g., at the 3' end) allow for the imaging group being located in direct proximity to the labeling site (UUA residue) of the labeled POI upon annealing of the imager strand with the POI- bound docking strand. The new Click-PAINT method was tested with two POIs. Cytoskeletal elements are an ideal starting point to validate SRM techniques as they result in defined filamentous patterns and the filament is highly enriched in individual proteins. The first POI (VimentinN116TAG-mOrange) was therefore a fusion protein com- prising at the N-terminus a mutant of cytoskeletal protein vimentin, wherein N116 was replaced by an amber codon (vimentinN116TAG) and at the C-terminus mOrange. The mOrange serves as a reference to check for specificity of the labeling using conven- tional wide-field microscopy. To test the sensitivity of the new Click-PAINT method, the second POI (GFPN149TAG- Nup153) was a protein of the nuclear pore complex, a much less abundant structure than the cytoskeleton. The nuclear pore complex is a ring-like structure built from about 30 different proteins and comprising 32 copies of the protein Nup153 which has an approximate size of 60 nm3. Thus, the density of potential labeling sites on Nup 153 is substantially lower than the density of potential labeling sites on cytoskeletal filaments. Specifically, the second pOI was a fusion protein comprising at the N-terminus mutated GFP, wherein N149 was replaced by an amber codon (GFPN149TAG) and at the C- terminus Nup153. Constructs for the expression of the target polypeptides were prepared, HEK293T cells were co-transfected with polynucleotides encoding tRNAPyl / NES-PylRSAF and either VimentinN116TAG-mOrange or GFPN149TAG-Nup153, and the transfected cells were cul- tured in UAA 2 using method (F) described herein. Click-PAINT labeling, imaging and image processing were carried out using methods (G) and (H) described herein. Figure 8c shows an SRM image generated using the Click-PAINT method described above and VimentinN116TAG-mOrange. Said image has a resolution that is clearly en- hances compared to the diffraction-limited imaging of the mOrange reference channel (see Figure 8b). Using GFPN149TAG-Nup153 as POI, the Click-PAINT method generated high-contrast, super-resolved images showing the typical circular appearance of nuclear pore com- plexs (see Figure 8d). Not all observed ring structures were closed because the cells also expressed wild-type Nup153 which cannot be labeled and competes for incorpora- tion into the nuclear core complexes with the GFPN149→2-Nup153 protein. Example 6: Amber suppression by PyIRSAF and NES-PyIRSAF in Baculovirus- based insect cell protein expression Sf21 cells were transduced with Bacmid DNA encoding tRNAPyl, mCherry-GFPY39TAG, and either PyIRSAF or NES-PyIRSAF, cultured with different concentrations (0, 10, 50, 100, 250, 500 or 1000 µM) of UAA 1 (BOC) and analyzed using methods (I) and (J) described herein. mCherry fluorescence of the cells indicated successful transduction with Bacmid DNA. GFP fluorescence of the cells indicated successful suppression of the amber stop co- don encoding amino acid position 39 of the GFP reporter gene by incorporation of UAA 1 at said position. Flow cytometric analysis showed an UAA 1-dose-dependent increase of mCherry- and GFP-fluorescent ("double positive") cells for both PyIRSAF and NES-PyIRSAF where the increase for NES-PyIRSAF-expressing cells was significantly more pronounced than in PylRSAF-expressing cells, indicating that NES-PylRSAF allowed for higher efficiency than PyIRSAF even at lower UAA 1 concentrations. See Figures 9, 10 and 11, and Ta- ble 1. Table 1: Relative size of fluorescent cell sub-populations in Sf21 cells transduced with Bacmid DNA encoding tRNAPyl, mCherry, GFPY39TAG and either PylRSAF or NES- PyIRSAF and incubated with different UAA 1 concentrations [Image disponible dans le document PDF, Image available in the PDF document] Abbreviations RS = aminoacyl tRNA synthetase BOC = Boc-L-Lys-OH = <semantics>N<annotation encoding="application / x-tex">N< / annotation>< / semantics>-<semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>-tert-butyloxycarbonyl-L-lysine (Figure 1a, compound 1) Crm1 = chromosomal region maintenance 1, also known as karyopherin exportin 1 dSTORM = direct stochastic optical reconstruction microscopy E. coli BL21(DE3)Al = E. coli strain B F- ompT gal dcm lon hsdSB(<semantics>rB<annotation encoding="application / x-tex">r_B< / annotation>< / semantics>-<semantics>mB<annotation encoding="application / x-tex">m_B< / annotation>< / semantics>-) <semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>(DE3 [lacl lacUV5-T7p07 ind1 sam7 nin5]) [malB+]K-12(λS) araB::T7RNAP-tetA FBS = fetal bovine serum FISH = fluorescence in situ hybridization GFP = green fluorescent protein Hoechst <semantics>33342=2′−(4−Ethoxyphenyl)−5−(4−methyl−1−piperazinyl)−2,5′−bi−1H−<annotation encoding="application / x-tex">33342 = 2'-(4-\text{Ethoxyphenyl})-5-(4-\text{methyl}-1-\text{piperazinyl})-2,5'-bi-1H-< / annotation>< / semantics> benzimidazole trihydrochloride IPTG = isopropyl <semantics>β<annotation encoding="application / x-tex">\beta< / annotation>< / semantics>-D-1-thiogalactopyranoside iRFP = infrared fluorescent protein NES = nuclear export signal NLS = nuclear localization signal 20 PBS = phosphate buffered saline PAINT = point accumulation for imaging in nanoscale topography PyIRS = pyrrolysyl tRNA synthetase PyIRSAF = mutant M. mazei pyrrolysyl tRNA synthetase comprising amino acid substi- tutions Y306A and Y384F PMSF = phenylmethylsulfonyl fluoride POI = polypeptide of interest, target polypeptide RP-HPLC = reversed phase high-performance liquid chromatography RT = room temperature TCEP = tris(2-carboxyethyl)phosphine TEV = Tobacco Etch Virus nuclear-inclusion-a endopeptidase tRNAPyl = tRNA that is acylated by a wild-type or modified PyIRS and has an anticodon that, for site-specific incorporation of the UAA into a POI, is preferably the reverse complement of a selector codon. (In the tRNAPyl used in the examples, the antico- don is CUA.) SPAAC = (copper-free) strain promoted alkyne-azide cycloaddition SPIEDAC = (copper-free) strain promoted inverse-electron-demand Diels-Alder cy- cloaddition SRM = super-resolution microscopy TB = Terrific Broth TCO* = N-<semantics>ε<annotation encoding="application / x-tex">\varepsilon< / annotation>< / semantics>-((trans-cyclooct-2-en-1-yloxy)carbonyl)-L-lysine (Figure 1a, compound 2) UAA = unnatural amino acid

Claims

<pat:ClaimStatement>CLAIMS< / pat:ClaimStatement> <pat:Claims com:id="claims"> <pat:Claim com:id="CLM-00001"> <pat:ClaimNumber>1< / pat:ClaimNumber> <pat:ClaimText>1. A modified archaeal pyrrolysyl tRNA synthetase, which is selected from the group consisting of: a modified Methanosarcina mazei pyrrolysyl tRNA synthetase comprising an unmodified Methanosarcina mazei pyrrolysyl tRNA synthetase modified by in- troduction of a nuclear export signal, and which retains pyrrolysyl tRNA synthe- tase activity, and a modified Methanosarcina barkeri pyrrolysyl tRNA synthetase comprising an unmodified Methanosarcina barkeri pyrrolysyl tRNA synthetase modified by in- troduction of a nuclear export signal, and which retains pyrrolysyl tRNA synthe- tase activity; wherein the unmodified Methanosarcina mazei pyrrolysyl tRNA synthetase comprises the sequence of SEQ ID NO:1 or 2, or a sequence at least 60% identical thereto over a full length thereof and that retains pyrrolysyl tRNA synthetase activity, and wherein the unmodified Methanosarcina barkeri pyrrolysyl tRNA synthetase comprises the sequence of SEQ ID NO:3, or a sequence at least 60% identical thereto over a full length thereof and that retains pyrrolysyl tRNA synthetase activity. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00002"> <pat:ClaimNumber>2< / pat:ClaimNumber> <pat:ClaimText>2. The modified archaeal pyrrolysyl tRNA synthetase of claim 1, wherein the unmodified Methanosarcina mazei pyrrolysyl tRNA synthetase com- prises the sequence of SEQ ID NO:1 or 2, or a sequence at least 70% identical thereto over a full length thereof and that retains pyrrolysyl tRNA synthetase activity, and wherein the unmodified Methanosarcina barkeri pyrrolysyl tRNA synthetase com- prises the sequence of SEQ ID NO:3, or a sequence at least 70% identical thereto over a full length thereof and that retains pyrrolysyl tRNA synthetase activity. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00003"> <pat:ClaimNumber>3< / pat:ClaimNumber> <pat:ClaimText>3. The modified archaeal pyrrolysyl tRNA synthetase of claim 1, wherein the unmodified Methanosarcina mazei pyrrolysyl tRNA synthetase com- prises the sequence of SEQ ID NO:1 or 2, or a sequence at least 80% identical thereto over a full length thereof and that retains pyrrolysyl tRNA synthetase activity, and wherein the unmodified Methanosarcina barkeri pyrrolysyl tRNA synthetase com- prises the sequence of SEQ ID NO:3, or a sequence at least 80% identical thereto over a full length thereof and that retains pyrrolysyl tRNA synthetase activity. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00004"> <pat:ClaimNumber>4< / pat:ClaimNumber> <pat:ClaimText>4. The modified archaeal pyrrolysyl tRNA synthetase of claim 1, wherein the unmodified Methanosarcina mazei pyrrolysyl tRNA synthetase com- prises the sequence of SEQ ID NO:1 or 2, or a sequence at least 90% identical thereto over a full length thereof and that retains pyrrolysyl tRNA synthetase activity, and wherein the unmodified Methanosarcina barkeri pyrrolysyl tRNA synthetase com- prises the sequence of SEQ ID NO:3, or a sequence at least 90% identical thereto over a full length thereof and that retains pyrrolysyl tRNA synthetase activity. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00005"> <pat:ClaimNumber>5< / pat:ClaimNumber> <pat:ClaimText>5. The modified archaeal pyrrolysyl tRNA synthetase of claim 1, wherein the unmodified Methanosarcina mazei pyrrolysyl tRNA synthetase com- prises the sequence of SEQ ID NO:1 or 2, or a sequence at least 95% identical thereto over a full length thereof and that retains pyrrolysyl tRNA synthetase activity, and wherein the unmodified Methanosarcina barkeri pyrrolysyl tRNA synthetase com- prises the sequence of SEQ ID NO:3, or a sequence at least 95% identical thereto over a full length thereof and that retains pyrrolysyl tRNA synthetase activity. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00006"> <pat:ClaimNumber>6< / pat:ClaimNumber> <pat:ClaimText>6. The modified archaeal pyrrolysyl tRNA synthetase of claim 1, wherein the unmodified Methanosarcina mazei pyrrolysyl tRNA synthetase com- prises the sequence of SEQ ID NO:1 or 2, and wherein the unmodified Methanosarcina barkeri pyrrolysyl tRNA synthetase com- prises the sequence of SEQ ID NO:

3. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00007"> <pat:ClaimNumber>7< / pat:ClaimNumber> <pat:ClaimText>7. The modified archaeal pyrrolysyl tRNA synthetase of any one of claims 1 to 6, which is the modified Methanosarcina mazei pyrrolysyl tRNA synthetase. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00008"> <pat:ClaimNumber>8< / pat:ClaimNumber> <pat:ClaimText>8. The modified archaeal pyrrolysyl tRNA synthetase of any one of claims 1 to 6, which is the modified Methanosarcina barkeri pyrrolysyl tRNA synthetase. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00009"> <pat:ClaimNumber>9< / pat:ClaimNumber> <pat:ClaimText>9. The modified pyrrolysyl tRNA synthetase of any one of claims 1 to 8, wherein the nuclear export signal comprises an amino acid sequence selected from the se- quences set forth in SEQ ID NOs:6-9. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00010"> <pat:ClaimNumber>10< / pat:ClaimNumber> <pat:ClaimText>10. A polynucleotide encoding the modified pyrrolysyl tRNA synthetase as defined in any one of claims 1 to 9. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00011"> <pat:ClaimNumber>11< / pat:ClaimNumber> <pat:ClaimText>11. The polynucleotide of claim 10, further encoding a tRNAPyl, wherein the tRNAPyl is a tRNA that can be acylated by the modified pyrrolysyl tRNA synthase encoded by polynucleotide as defined in claim 10. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00012"> <pat:ClaimNumber>12< / pat:ClaimNumber> <pat:ClaimText>12. A combination of polynucleotides comprising at least one polynucleotide of claim 10 and at least one polynucleotide encoding a tRNAPyl, wherein the tRNAPyl is a tRNA that can be acylated by the modified pyrrolysyl tRNA synthase encoded by polynucleotide of claim 10. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00013"> <pat:ClaimNumber>13< / pat:ClaimNumber> <pat:ClaimText>13. The polynucleotide of claim 10 or claim 11 or the combination of polynucleotides of claim 12, wherein the anticodon of the tRNAPyl is the reverse complement of a co- don that is selected from stop codons, four base codons and rare codons. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00014"> <pat:ClaimNumber>14< / pat:ClaimNumber> <pat:ClaimText>14. An isolated eukaryotic cell comprising: (a) a polynucleotide sequence encoding the modified pyrrolysyl tRNA synthetase as defined in any one of claims 1 to 9, and (b) a tRNA that can be acylated by the pyrrolysyl tRNA synthase encoded by the sequence of (a), or a polynucleotide sequence encoding such tRNA. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00015"> <pat:ClaimNumber>15< / pat:ClaimNumber> <pat:ClaimText>15. The isolated eukaryotic cell of claim 14, wherein the cell is a mammalian cell. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00016"> <pat:ClaimNumber>16< / pat:ClaimNumber> <pat:ClaimText>16. A method for preparing a target polypeptide comprising one or more than one un- natural amino acid residue, wherein the method comprises: (a) providing a eukaryotic cell comprising: (i) the modified pyrrolysyl tRNA synthetase as defined in any one of claims 1 to 9, (ii) a tRNAPyl, (iii) an unnatural amino acid or a salt thereof, and (iv) a polynucleotide encoding the target polypeptide, wherein any position of the target polypeptide occupied by an unnatural amino acid residue is encoded by a codon that is the reverse complement of the anticodon comprised by the tRNAPyl; and wherein the pyrrolysyl tRNA synthetase (i) is capable of acylating the tRNA by the tRNA by the tRNA by the tRNA by the tRNA by the tRNA by the tRNA by the tRNA by the tRNA by the tRNA by the tRNA by the tRNA by the tRNA by (ii) with the unnatural amino acid or salt (iii); and (b) allowing for translation of the polynucleotide (iv) by the eukaryotic cell, thereby producing the target polypeptide. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00017"> <pat:ClaimNumber>17< / pat:ClaimNumber> <pat:ClaimText>17. A method for preparing a polypeptide conjugate comprising: (a) preparing a target polypeptide comprising one or more than one unnatural amino acid residue using the method of claim 16; and (b) reacting the target polypeptide with one or more than one conjugation partner molecule such that the conjugation partner molecules bind covalently to the unnatural amino acid residue(s) of the target polypeptide. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00018"> <pat:ClaimNumber>18< / pat:ClaimNumber> <pat:ClaimText>18. A kit comprising at least one unnatural amino acid, or a salt thereof and: (a) the polynucleotide of any one of claims 10, 11 and 13, (b) the combination of polynucleotides of claim 12 or 13, or (c) the isolated eukaryotic cell of claim 14 or 15; wherein the modified archaeal pyrrolysyl tRNA synthetase is capable of acylating the tRNAPyl with the unnatural amino acid or salt thereof. < / pat:ClaimText> < / pat:Claim> < / pat:Claims>